Shan Shu-ou, Walter Peter
Howard Hughes Medical Institute and Department of Biochemistry and Biophysics, University of California, San Francisco, California 94143-2200, USA.
Biochemistry. 2005 Apr 26;44(16):6214-22. doi: 10.1021/bi0500980.
In signal recognition particle (SRP)-dependent targeting of proteins to the bacterial plasma membrane, two GTPases, Ffh (the SRP GTPase) and FtsY (the receptor GTPase), form a complex in which both proteins reciprocally stimulate each other's GTPase activities. We mutated Asp251 in the Ffh active site to Asn (D251N), converting Ffh to a xanthosine 5'-triphosphate (XTP)-specific protein as has been observed in many other GTPases. Unexpectedly, mutant SRP(D251N) is severely compromised in the formation of an active SRP.FtsY complex when bound with cognate XTP, and even more surprisingly, mutant SRP(D251N) works better when bound with noncognate GTP. These paradoxical results are explained by a model in which Ffh Asp251 forms a bidentate interaction with not only the bound GTP but also the receptor FtsY across the dimer interface. These interactions form part of the network that seals the lateral entrance to the composite active site at the dimer interface, thereby ensuring the electrostatic and/or structural integrity of the active site and contributing to the formation of an active SRP.FtsY complex.
在信号识别颗粒（SRP）依赖的蛋白质靶向细菌质膜过程中，两种GTP酶，即Ffh（SRP GTP酶）和FtsY（受体GTP酶），形成一个复合物，其中两种蛋白质相互刺激对方的GTP酶活性。我们将Ffh活性位点中的Asp251突变为Asn（D251N），使Ffh转变为一种对5'-三磷酸黄苷（XTP）具有特异性的蛋白质，这在许多其他GTP酶中都有观察到。出乎意料的是，突变型SRP（D251N）在与同源XTP结合时，活性SRP.FtsY复合物的形成严重受损，更令人惊讶的是，突变型SRP（D251N）与非同源GTP结合时效果更好。这些矛盾的结果可以用一个模型来解释，即Ffh Asp251不仅与结合的GTP形成双齿相互作用，还通过二聚体界面与受体FtsY形成双齿相互作用。这些相互作用构成了网络的一部分，该网络封闭了二聚体界面处复合活性位点的侧向入口，从而确保活性位点的静电和/或结构完整性，并有助于形成活性SRP.FtsY复合物。
